The primary aim of Project 2 was to use unbiased high throughput screening (HTS) of small molecule libraries allied to more targeted approaches to identify novel compounds that would protect and mitigate against radiation damage, primarily to the immunohematopoietic system but also other tissues. The theory was that active compounds would possess a molecular and/or chemical signature that could be identified and optimized to benefit the development of superior agents for Stockpiling in case of a radiologic disaster. We have shown that tetracyclines, quinolones, and cyclopiazonic acid can act as radiation mitigators, independent of any anti-microbial action. Furthermore, they share a common metal-binding pharmacophore. The importance of this substructure for mitigation will be studied in the next funding period. Other active mitigators included purine nucleosides. We intend to explore this avenue further using new compounds that are specific for differentially expressed subtypes of adenosine receptors so as to minimize possible mutually antagonistic actions and side effects resulting from the use of these agents. Linoleate and other polyunsaturated fatty acids also showed mitigating activity, as did several other agents that are directed to Toll-like receptors or are products of these pathways. This link will be investigated furter in collaboration with Project 3. Compounds from the chemically defined libraries that were positive in our screens will be investigated further along with Project 1, which showed that two ofthe compounds were positive in yeast DEL HTS assays and in vivo as mitigators. Our working hypothesis is that radiation initiates continuing waves of integrated molecular and cellular responses that are aimed at tissue regeneration and that multiple mitigators may be needed, perhaps given at different times, to rebalance tissue homeostasis. This will require mechanistic knowledge of how these mitigators work. We have shown effects ranging from DNA repair to stimulating hematopoiesis to long-term animal survival. In vitro assays will be extended to include novel epithelial and stem cell assays developed through the Pilot Project mechanism and in vivo assays will be extended to include better coverage of effects of sublethal damage and stem cell recovery.